1. Field of the Invention
The present invention relates generally to microcalorimeters and more specifically to features that improve the performance of microcalorimeters, especially isothermal titration calorimeters.
2. Background
Microcalorimeters are broadly utilized in fields of biochemistry, pharmacology, cell biology, and others. Calorimetry provides a direct method for measuring changes in thermodynamic properties of biological macromolecules. Microcalorimeters are typically two cell instruments in which properties of a dilute solution of test substance in an aqueous buffer in a sample cell are continuously compared to an equal quantity of aqueous buffer in a reference cell. Measured differences between the properties of the two cells, such as temperature or heat flow, are attributed to the presence of the test substance in the sample cell.
One type of microcalorimeter is an isothermal titration calorimeter. The isothermal titration calorimeter (ITC) is a differential device, but operates at a fixed temperature and pressure while the liquid in the sample cell is continuously stirred. The most popular application for titration calorimetry is in the characterization of the thermodynamics of molecular interactions. In this application, a dilute solution of a test substance (e.g., a protein) is placed in the sample cell and, at various times, small volumes of a second dilute solution containing a ligand, which binds to the test substance, are injected into the sample cell. The instrument measures the heat which is evolved or absorbed as a result of the binding of the newly-introduced ligand to the test substance. From results of multiple-injection experiments, properties, such as, the Gibbs energy, the association constant, the enthalpy and entropy changes, and the stoichiometry of binding, may be determined for a particular pairing between the test substance and the ligand.
While currently utilized ITCs provide reliable binding data results, their widespread utilization in the early stages of drug development have been limited by several factors: the relatively high amounts of protein required to perform a binding determination (e.g., about 0.1 milligram (mg) to about 1.0 mg of a protein), the time required to perform the measurement, and the complexity of using conventional ITCs. Due to the extremely high costs of biological substances used in research, there is a need to reduce the amount of biological substance used for each experiment. A reduction in the amount of the biological substance used in a calorimeter experiment, will require a more accurate, sensitive, and reliable titration calorimeter than what is currently available.
Furthermore, gathering binding data utilizing prior art ITCs require extensive preparation and skill by the practitioner. For example, using prior art ITCs, the reference and sample cells are first filled respectively with the reference substance and sample substance via a corresponding cell stem. Next, a syringe of the ITC is filled with a titrant. Then a needle of the ITC is placed in the sample cell via a cell stem leading to the sample cell while the syringe fits into a holder on the ITC enabling the syringe to rotate around its axis. Subsequently, the syringe is aligned with the sample cell so that the needle does not touch either the cell stem or the sample cell. Then, the syringe is connected to a stirring motor and a linear activator of the ITC, wherein the stirring motor and the linear activator must also be aligned with the sample cell.
As would be appreciated by a reading of the above-described prior art procedure, utilizing prior art ITCs, the quality of binding measurements performed with these prior art ITCs depends heavily of the operator's skills and experience, and involves a considerable amount of preparation time.
More recently developed prior art ITCs have attempted to simplify the preparatory procedures described above. For example, in one such prior art ITC, which is partly shown in FIG. 1 as ITC 100, a syringe 102, a syringe holder 104, and a linear actuator 106, which actuates syringe 102's plunger, are integrated into a single unit referred to as an automatic pipette. ITC 100 further comprises a stirring mechanism comprising a stirring motor 108, which is attached to calorimeter body 110. ITC 100 also comprises an inner magnet couple 112 located around syringe 102, and an outer magnet couple 114 located on calorimeter body 110 in close proximity to stirring motor 108. The rotation from stirring motor 108 to syringe 102 is transferred via magnet couplings 112 and 114. Attached to syringe 102 is a needle 116 and a paddle 118. The needle 116 is arranged to be inserted into a sample cell 120 via a cell stem 122 for performing ITC experiments. For reference purposes, ITC 100, also comprises a reference cell, not shown, in communication with the ambient atmosphere via a reference cell stem.
The prior art design discussed above and depicted in FIG. 1 has certain limitations. For example, since the magnet coupling is a soft/flexible transmission, it is prone to resonant vibration of the stirrer at certain rotation speeds and accelerations, which negatively affects the instrument's sensitivity. The resonant vibration can be reduced by either employing a less sensitive feedback mechanism controlling the rotation speed (which leads to less stable rotation speed), or by lowering the rotation speed. However, less stable rotation speed also reduces the ITC's sensitivity, while lower rotation speed impedes proper mixing of reagents which reduces the ITC's accuracy.
Another limitation of the prior art design is that the stirring motor and the magnet coupling are placed closely to the sensitive measuring unit of the instrument and generates a substantial alternating magnetic field that produces electric noise which negatively affects the operation of the ITC's sensitive electronic circuitry. Since the ITC's sensors process signals of approximately 10−9 volts, and the noise generated by the motor and the magnetic coupling is a reciprocal of the distance between the sensor and the source of the noise, further improvements in the performance characteristics of this ITC design become increasingly challenging. As stated earlier, one of the underlying factors affecting the design of new microcalorimeters is the need to reduce the amount of biological substance used for each experiment. This requires smaller sample cells and shorter cell stems which in turn leads to, smaller distances between the cell sensor and the motor and magnetic coupling (source of electric noise), which limits the instrument's sensitivity.
The invention described herein is aimed to improve the aforementioned characteristics and use of prior art ITCs such that the sensitivity of the ITC is improved, the amount of biological substance necessary for testing is reduced, the reliability of the results generated by the ITC is improved, and use of the ITC is eased.